This chapter discusses molecular genetics of Archaea. Gene transfer systems currently exist for species within all three physiological groups of archaea, halophiles, methanogens, and nonmethanogenic hyperthermophiles. The chapter reviews the development of these systems. Colonization of methanogenic and nonmethanogenic hyperthermophiles on solidified medium is equally problematic, as agar is rapidly dehydrated at high temperatures, especially at the concentrations required for it to remain solidified. Therefore, gellan gum (Gelrite) is used as the solidifying agent for growth of thermophiles and hyperthermophiles, which are incubated in a plastic bag or anaerobe jar to minimize dehydration. Haloarchaea are transformed via polyethylene glycol (PEG) mediated transformation, which was first described in Haloferax volcanii. Gene disruption is required to identify and confirm the function of genes within the archaea. Random mutagenesis using chemical and UV radiation has been successfully used for H. volcanii, Methanococcus voltae, Methanococcus maripaludis, and Pyrococcus abyssi. Progress in the development of methodologies for archaeal genetics has rapidly accelerated in the past decade. Additional methods are currently under development for all three archaeal phyla, including additional systems for markerless exchange, gene expression, topological mapping, protein tagging and expression, as well as others. The choice of archaeal genomes for sequencing is now largely driven by the availability of genetic systems, which at present include complete genomes of the halophiles Haloarcula marismortui and Halobacterium sp.

Gene disruption methods that are used in archaeal genetics. (a) Direct replacement of a gene with a selectable marker occurs by recombination between linear DNA flanked by regions of the target gene and the wild-type chromosomal gene. (b) The “pop-in pop-out” method uses circular DNA and selection for transformation to uracil prototrophy using a ura- strain (17, 85, 96). Recombinants that have lost the plasmid are counter-selected using 5-fluoroorotic acid (5-FOA), which inhibits growth of ura+ cells. Deletion mutants must be screened by Southern hybridization. (c) A variant of the “pop-in pop-out” method for gene deletion utilizes a genetic marker for gene disruption that allows direct selection (4). (d) Another variant used for generating point mutations employs gene replacement with a ura marker and subsequent replacement of the ura disrupted target gene with a gene containing the desired point mutation (85). Mutants with the desired point mutation are counter-selected with 5-FOA. Reprinted from Nature Reviews Genetics (3) with permission of the publisher.

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10.1128/9781555815516/f0470-01.gif

Figure 2.

Gene disruption methods that are used in archaeal genetics. (a) Direct replacement of a gene with a selectable marker occurs by recombination between linear DNA flanked by regions of the target gene and the wild-type chromosomal gene. (b) The “pop-in pop-out” method uses circular DNA and selection for transformation to uracil prototrophy using a ura- strain (17, 85, 96). Recombinants that have lost the plasmid are counter-selected using 5-fluoroorotic acid (5-FOA), which inhibits growth of ura+ cells. Deletion mutants must be screened by Southern hybridization. (c) A variant of the “pop-in pop-out” method for gene deletion utilizes a genetic marker for gene disruption that allows direct selection (4). (d) Another variant used for generating point mutations employs gene replacement with a ura marker and subsequent replacement of the ura disrupted target gene with a gene containing the desired point mutation (85). Mutants with the desired point mutation are counter-selected with 5-FOA. Reprinted from Nature Reviews Genetics (3) with permission of the publisher.

Markerless disruption method that employs Flp recombinase. An artificial operon that expresses puromycin N-acetyl-transferase (pac) and hypoxanthine phosphoribosyltransferase (htp) is flanked by Flp recombinase recognition sites (RP1 and RP2) and regions homologous to the target gene. The linear DNA is transformed into an M. acetivorans Δhpt strain that is resistant to 8-aza-2,6-diamino-purine (8-ADP). The target gene is replaced by homologous recombination, and recombinants are selected by resistance to puromycin. The deletion mutant is subsequently transformed with the nonreplicating plasmid pMR55 encoding Flp recombinase, which removes the pac-hpt operon by site-specific recombination between RP1 and RP2. Reprinted from Current Opinion in Microbiology (94) with permission of the publisher.

10.1128/9781555815516/f0472-01_thmb.gif

10.1128/9781555815516/f0472-01.gif

Figure 3.

Markerless disruption method that employs Flp recombinase. An artificial operon that expresses puromycin N-acetyl-transferase (pac) and hypoxanthine phosphoribosyltransferase (htp) is flanked by Flp recombinase recognition sites (RP1 and RP2) and regions homologous to the target gene. The linear DNA is transformed into an M. acetivorans Δhpt strain that is resistant to 8-aza-2,6-diamino-purine (8-ADP). The target gene is replaced by homologous recombination, and recombinants are selected by resistance to puromycin. The deletion mutant is subsequently transformed with the nonreplicating plasmid pMR55 encoding Flp recombinase, which removes the pac-hpt operon by site-specific recombination between RP1 and RP2. Reprinted from Current Opinion in Microbiology (94) with permission of the publisher.

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